Growth of foreign cells after conditional and selective destruction of fetal host cells

ABSTRACT

Foreign cells can be grown in fetal non-mammalian hosts for the production of transplant organs and tissues, the development of new therapeutic agents, and the production of biological factors and drugs. Tissue-specific injury to fetal host target cells is carried without substantial injury to the maternal host or foreign cells, providing an environment in which the injured tissue can be regenerated with the foreign cells.

This application is a continuation of pending U.S. Ser. No. 10/527,587,filed Feb. 21, 2006, which is a national stage application of PCTapplication PCT/US2003/02951, filed Sep. 17, 2003, which claims thebenefit of provisional application Ser. No. 60/411,790, filed Sep. 19,2002. These applications are incorporated herein in their entireties.

This application incorporates by reference the contents of a 12 kb textfile created on Nov. 2, 2009 and named “000241_(—)00013_Seq_List.txt,”which is the sequence listing for this application.

This invention was made with government funds from ATP grant no.70NANB0H3008 from NIST, Department of Commerce. The government retainscertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the growth and differentiation of foreign cellswithin a mammalian host. In particular, the invention relates tochimeric mammals that can be used to develop new drugs and vaccines, aswell as to produce factors, drugs, and tissues for transplantation.

BACKGROUND OF THE INVENTION

Efforts to produce cells in vitro have met with limited success. Whileembryonic stem cells can be expanded indefinitely, it is difficult toexpand differentiated cells. Moreover, it is currently notcost-effective to produce differentiated cells from stem cells in vitro.

The growth of foreign cells within an animal would provide substantialvalue in biotechnology. The production, expansion and isolation of cellsusing a non-human mammalian host would provide cells for infusion andtransplantation, the production of drugs and factors for therapy, cellsfor tissue engineering and assays. The production of animals that arechimeric, and contain foreign cells would be useful for transplantation,models of disease, and for the functional assessment of a transgene.

Two factors make it challenging to grow foreign cells in animals,however. First, the foreign cells normally would be rejected by the hostanimal. Second, the foreign cells would need to compete with the nativecells of the animal.

Cells have been grown in congenitally immune deficient animals. Forexample, human lymphocytes have been grown within SCID (severe combinedimmune deficiency) mice (1). These mice normally have a deficiency of Band T cells. However, the human lymphocytes are not appropriatelyfunctional and do not provide a normal immune response (2, 3).

Transgenic mice have been used to enhance engraftment with foreigncells. Rhim and Brinster produced transgenic mice with a defectiveurokinase plasminogen activator gene controlled by an albumin promoter.The native hepatocytes in these mice were defective and did not survivelong. The defective hepatocytes were eventually replaced when foreignhepatocytes, including rat hepatocytes were injected (4). This model isnot practical, however. Mice cannot be used as a source of donor organs.In addition, the pups had hypofibrinogenemia and usually died ofneonatal hemorrhage (5).

SCID mice that are homozygous for urokinase plasminogen activator (uPA)have been engrafted with human hepatocytes (6). Due to the death of themouse hepatocytes, the homozygous mice are difficult to keep alive.Heterozygous mice must be bred, and the homozygous offspringtransplanted right after birth. The mice often die of liver failurebefore the human hepatocytes provide support. Because they lack afunctional immune system, however this model has limited value for thedevelopment of vaccines.

Braun et al. used adult transgenic mice containing the suicide genethymidine kinase to enhance engraftment with foreign cells (7). Thethymidine kinase was under control of an albumin promoter and wasexpressed in the hepatocytes. The hepatocytes were normal until theprodrug gancyclovir was administered to adult mice. Most of thehepatocytes then died off, leading to regeneration with new hepatocytes.This system was an improvement over the uPA mouse model, because itpermits controlled killing and turnover of the hepatocytes. But whileengraftment was enhanced, overall survival was not generally improved.Following hepatic necrosis, most mice did not survive long enough toallow the differentiation and organization of the new hepatocytes. Thesmall size of the mouse also limits the application of this system,because chimeric livers could not be produced for human transplantation.

Foreign cells have been infused into fetal animals, leading to limitedengraftment. For example, limited engraftment of hematopoietic cells hasbeen demonstrated in fetal sheep and monkeys (8) and by infusion intofetal mice, sheep, and pigs (9, 10, 11). Infusion of human hepatocytesor stem cells into fetal pigs has resulted in only limited engraftment(12). The intrauterine environment is favorable to engraftment withforeign cells, and the host naturally develops immune tolerance to thecells (13). The uterine environment is also naturally sterile. However,engraftment of foreign cells is very limited due to competition with thenative host cells.

While transgenic mice can be readily produced to study diseases relatedto a specific gene, it is not practical to produce large animals. Forexample, pigs are several thousand times larger than mice and theirgenerational time is about 10 times as long. To date, there has onlybeen one herd of transgenic pigs produced for the study of humandisease, retinitis pigmentosa (Petters et al., Nature Biotechnology 15,965, 1997).

Thus, there is a need, in the art for methods of engrafting foreigncells in fetal host animals.

DESCRIPTION OF RELATED PATENTS

U.S. Pat. No. 5,672,346 to Srour et al. teaches the infusion ofhematopoietic stem cells into fetal non-human mammals leading to limitedengraftment of hematopoietic cells. It does not provide a method for theelimination of native cells in the fetus.

U.S. Pat. No. 5,411,749 to Mayo et al. teaches the implantation of humanlymphoid tissue into a mouse that is genetically immunodeficient for Tand B lymphocytes. It does not provide for conditional elimination ofnative cells. The engrafted lymphocytes have limited function. Forexample, they are unable to provide a primary immune response to anantigen.

U.S. Pat. No. 5,698,767 to Wilson and Mosier teaches the implantation ofhuman leukocytes into a SCID mouse, genetically immune deficient of Tand B lymphocytes. The engrafted lymphocytes have limited function.

U.S. Pat. No. 6,211,429, “Complete oocyte activation using anoocyte-modifying agent and a reducing agent,” to Z. Machaty and R. S.Prather, teaches the development of transgenic animals, including pigs,cows, sheep, mice, dogs, mice, and horses using nuclear transfer ofcells transfected with a gene for growth hormone, placental lactogen,etc. It does not specifically teach the production of large transgenicanimals with suicide gene or the induction of selective fetal tissueinjury.

U.S. Pat. No. 6,147,276, “Quiescent cell populations for nucleartransfer in the production of non-human mammals and non-human mammalianembryos,” to K. H. S. Campbell and I. Wilmut teaches the production oftransgenic animals using nuclear transfer. It does not specificallyteach the production of transgenic animals containing suicide genes orthe selective and conditional injury to fetal tissues.

U.S. Pat. No. 6,291,740, “Transgenic Animals,” to R. D. Bremel, A. W. S.Chan, and J. C. Burns, teaches the production of transgenic animalsusing perivitelline space injection of the transgene. It does not teachthe use of a suicide gene for selective and conditional injury of fetaltissue.

SUMMARY OF THE INVENTION

One embodiment of the invention provides methods for enhanced growth offoreign cells within non-human mammals. The methods employ conditionaland controlled reduction of select cells within a tissue of a fetalnon-human mammal, followed by regeneration of the tissue with theforeign cells. The destruction of the fetal cells does not affect thecorresponding cells in the maternal host or the foreign regeneratingcells. The destruction of fetal cells is specific for the cells that arebeing replaced during tissue regeneration.

Another embodiment of the invention provides viable fetal non-humanmammals with selective injury to target tissues, as well as maternalhosts comprising such fetuses. The fetuses are in a condition suitablefor infusion with foreign cells, which can regenerate the injured targettissues. In some embodiments, fetal non-human mammals that contain asuicide transgene product produced in a target tissue have been exposedto the appropriate prodrug. In other embodiments, fetal non-humanmammals contain suicide transgene products in all tissues and have beenexposed to liposomes or immunoliposomes specific for the target tissue,which also can deliver the appropriate prodrug.

In another embodiment, the invention includes compositions and methodsfor enriching a population of cells from a chimeric animal for theforeign cells, by adding a prodrug that kills native cells expressing asuicide transgene.

The invention also provides fetal non-human mammals having a selectcellular injury induced within the uterus of a normal maternal host. Thecellular injury in the fetus is conditional and can be induced at thediscretion of the user.

The invention provides methods for the growth of cells within fetalnon-human mammals that have value for organ and cellulartransplantation, production of factors by the foreign cells, and modelsof disease and physiology for development of new therapeutic agents.

The invention also provides mammals that express a suicide transgeneproduct under the control of either a tissue-specific or a universalpromoter, as well as methods for separating expanded foreign cells fromnative cells in a suspension.

In another embodiment of the invention, replacement cells are placedwithin a host animal during fetal development. The cells engraft andestablish tolerance of the host animal to the replacement cells. Thenative host cells are subsequently depleted in a conditional andselective manner. This can be accomplished using transgenic animalsexpressing a suicide gene that would be injected with replacement cellsin utero. Prodrug is be administered in a manner to conditionally andselectively destroy the native host cells. The prodrug can beadministered on multiple occasions and administered after birth of theanimals.

In yet another embodiment of the invention, select host cells arereplaced with abnormal cells to produce models of human disease. Thereplacement cells can be from the same species as the host animal. Inthis case, the replacement cells can be transfected with the abnormalgene or they can have the test gene deleted (“knocked out”). Thereplacement cells can be from the same or from a different species, suchas from a human with a congenital enzyme abnormality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Western blot analysis of TK expression under the control ofdifferent promoters.

FIG. 2. Genetic constructs for suicide transgenes.

DETAILED DESCRIPTION Overview

To achieve significant replacement of native cells in a host animal withforeign cells, it is preferable to selectively eliminate native cells ofthe host animal in a controlled fashion without harming the foreignengrafting cells. In addition, there should be physiologic support forthe host animal during the period between elimination of native cellsand engraftment with the foreign replacement cells. This inventionachieves these goals by providing methods for selective and/orconditional destruction of cells in tissues of a fetal host withoutsubstantial destruction of cells in the maternal host mammal or injuryof the foreign replacement cells (i.e., less than about 50, 45, 40, 35,30, 25, 20, 15, 10, or 5% loss of maternal or foreign replacementcells). The maternal host mammal provides physiologic support while theforeign replacement cells engraft and regenerate the fetal tissue. Theresulting chimeric or hybrid tissue is useful, for example, as a sourceof cells and tissue for transplantation, as well as for the productionof factors and drugs for therapeutic purposes. The chimeric fetal hostis useful, e.g., for the development of drugs and vaccines.

The invention also provides human cells for toxicology testing andresearch purposes. For example, human hepatocytes are in very shortsupply. Most available viable human cadavers are used liver transplants,and it is not possible to grow non-transformed human hepatocytes inculture. By growing and isolating human hepatocytes from hybrid pigs asdisclosed herein, the invention can provide hepatocytes for research andfor toxicology studies.

Methods of the invention provide multiple advantages for expanding anddifferentiating foreign replacement cells, including human cells, withina fetus of a non-human host mammal. A “fetus” is the unborn offspring ofa viviparous animal. A fetus is distinguished from an embryo by thepresence of the beginnings of all major structures (14). The fetalenvironment allows for growth of foreign replacement cells withoutimmune rejection. The fetal environment also is ideal for the growth ofnew cells, providing growth factors needed for expansion of tissues andnutritional and physiological support for the temporary deficiencycaused by the depletion of the target cells. The absence of the cellulardeficiency in the maternal host is advantageous, allowing the fetuses todevelop to term.

One or more tissues in the fetal host is injured and partially orcompletely eliminated, making the tissue receptive to regeneration withforeign replacement cells. Cells of most tissues can be regeneratedusing methods of the present invention. A “tissue” comprises a group ofsimilarly specialized cells that perform a common function (e.g., liver,hematopoietic, endothelial, neural, epithelial, retinal, pigmentepithelial, myocardial, skeletal muscle, smooth muscle, lung, intestine,kidney, endocrine, cartilage, or bone cells). A tissue can be eithersolid (e.g., liver) or dispersed (e.g., blood). Tissues that can beregenerated according to methods of the invention include, but are notlimited to, liver, bone marrow, neural tissue, smooth muscle, skeletalmuscle, cardiac muscle, skin, retinal pigment epithelium, pancreaticislets, endothelium, thymus, including thymic epithelium, lymphocytes,urogenital epithelium, renal tissues, stem cells and progenitor cells,pulmonary epithelium, bronchial epithelium, breast tissue, cartilage,bone, intestine, and intestinal epithelium.

Replacing native tissue cells with foreign tissue cells, such ashepatocytes, is useful for many purposes. The new hepatocytes canproduce therapeutic proteins. A chimeric liver comprising humanhepatocytes is useful as a source of cells for transplantation. Thechimeric liver itself is valuable for transplantation and also providesa model system for development of therapies for diseases such ashepatitis. Human hepatocytes can be isolated from multiple pigs andinfused into patients with chronic liver failure or congenital liverabnormalities.

A chimeric animal whose bone marrow cells are replaced with foreign bonemarrow cells (e.g., human hematopoietic cells) can provide a costeffective bioreactor for producing human cells for transfusion, such asred blood cells, granulocytes, lymphocytes, dendritic cells,macrophages, megakaryocytes, platelets, etc. The chimeric animal alsocan be used as a model system for modulating the human immune responseto antigens and for the development of new vaccines.

Foreign neural cells can be produced in host mammals for transplantationinto patients with neurodegenerative diseases or spinal cord injuries.

Replacement of fetal host skin epithelium with foreign epithelium,including human epithelium, is a cost-effective method of producing skinfor treatment of wounds, ulcers, and burns, for toxicology studies, andfor the development of drugs and vaccines for pathogens that selectivelyinfect human skin, such as the papilloma virus. Epithelial cells arealso important in development of tolerance in the thymus. Thedevelopment of human urogenital epithelium can be used in thereconstruction and transplantation of urinary bladders, urethras, andureters.

Retinal pigment epithelium, such as human retinal pigment epithelium,produced in the fetal host mammal can be used to treat maculardegeneration.

The replacement of pancreatic islets with foreign islets, includinghuman islets, is valuable for transplantation into patients withdiabetes.

Endothelial cells express many of the transplant antigens responsiblefor rejection. Thus, the replacement of fetal host mammal endothelialcells with foreign endothelial cells would be valuable for thetransplantation of vascular organs such as hearts, lungs, kidneys, etc.

The replacement of host renal epithelial cells with foreign replacementcells such as human renal cells would produce better xenografts fortransplantation, for production of human erythropoietin, and for controlof blood pressure.

The growth of foreign stem cells and progenitor cells in place of thecorresponding fetal host cells would be useful as a cost-effectivebioreactor for the production of multipotential stem cells for stem celltherapy.

The replacement of pulmonary and bronchial epithelium with foreignreplacement cells such as human pulmonary and bronchial epithelium wouldproduce valuable organ transplants for the treatment of disorders suchas chronic pulmonary failure and cystic fibrosis.

The replacement of breast epithelial cells with foreign replacementcells would be valuable for the production of therapeutic factors. Theforeign replacement cells would be transfected for the desired protein.The protein could be easily harvested from the milk of a chimericanimal.

The growth of foreign cartilage and bone cells in host mammals would beuseful for reconstructive surgery, as well as for the development of newdrugs to treat disorders such as arthritis.

Human intestinal epithelial xenografts can be grown and used to treatcongenital short gut syndrome and chronic intestinal diseases such asCrohn's Disease.

Transgenic Animals and Host Animals

Fetal and maternal host animals preferably are non-human mammals, suchas non-human primates, artiodactyls, carnivores, rodents, or lagomorphs.Large mammals, such as pigs, sheep, cows, or non-human primates, areuseful for producing organs or large numbers of cells suitable for humantransplantation. Non-human primates are suitable from the standpoint oforgan function and similarity to human cells. Amino acid sequencing ofproteins typically demonstrate 90 to 98% homology with humans. Whilesome of the lower primates, such as lemurs, have short gestation periods(132-134 days), the higher primates (chimpanzees, gorillas) havegestation periods approximating that of humans (267 days).

The artiodactyls (even-toed ungulates) include several domesticatedanimals such as pigs, sheep, goats, and cows. Many of their organs aresimilar to those of humans and have been shown to function within humansor non-human primates. The gestation periods vary between the members ofthis order. Pigs have a period of 114 days. Sheep have a period of 145days. Cows have a gestation period of 282 days. Pigs, sheep, and cowsoffer specific advantages as a host animal. Pigs have large litters andshort gestation periods. Fetal lambs are easy to inject with foreignreplacement cells. Cows produce large offspring providing the potentialfor greater expansion. Also cells infused into one calf may circulate tothe other calves through shared placental circulation. The pig is apreferred host.

The carnivores, including dogs, cats, etc., have several features thatare potentially advantageous. Many have short gestation periods (catsabout 65 days, dogs about 63 days), and their newborn are relativelywell developed. Cats and dogs are often used as models of physiology andtransplantation (22, 23).

Rodents, including rats, mice, etc., are useful for engraftment withforeign replacement cells because of their short gestation periods andrapid growth to maturity. For example, rats have a gestation period ofonly 21 days and grow to maturity in only 6 weeks. It is relatively easyto produce transgenic rodent animals, including knockout animals withdeletion of a gene. Lagomorphs, which include rabbits and hares, sharewith rodents a very short gestation period and short maturation periods.Their larger size, however, make these animals better for producinglarger organs and achieving greater expansion of the foreign replacementcells than the rodents.

Fetal hosts can be obtained by mating a transgenic male animal with anon-transgenic female animal. A “transgenic animal” comprises a“transgene,” i.e., a gene that did not original in the transgenic animal(a foreign gene). A transgene may or may not be integrated into DNA of ahost cell. The transgene preferably is integrated into the DNA of thetransgenic animal's germ cells.

Transgenic animals can be made by any method known in the art, includingnuclear transfer, intracellular sperm injection, and perivitelline spaceinjection (33, 34). Either the male or female parent can carry thetransgene and can be either heterozygous or homozygous. In oneembodiment, a non-transgenic female mammal is bred with a transgenicmale mammal containing a suicide transgene whose expression iscontrolled by a tissue-specific promoter. A transgenic male should befertile and produce sperm with the suicide transgene in the genome.Preferably, the male is homozygous for the suicide transgene, so thatall of the fetuses would then be heterozygous for the suicide gene.Heterozygous males can be used, though only one half of the fetuseswould contain the suicide transgene.

If the prodrug is to be administered systemically to the maternal host,such as by injection into a sow, then a male transgenic animal should bebred with a wild-type, non-transgenic maternal host. Then, only thefetuses would express the suicide gene product. Optimally, a transgenicmale would be homozygous, expressing the gene on both somaticchromosomes. Then all of the fetuses would carry the suicide gene in aheterozygous manner. The transgenic animal could be heterozygous aswell, but only a portion of the fetuses would express the suicide gene.

For example, to grow human liver cells (hepatocytes) in pig livers, anon-transgenic female pig (gilt or sow) would be bred with a transgenicmale pig (boar) a suicide gene controlled by a liver specific promotersuch as an albumin or an α-fetoprotein promoter. The fetal pigs thenexpress the suicide gene product in the target tissue, but the gilt orsow does not express it. Other tissue-specific promoters are known inthe art and can be used as appropriate, such as the breast specificpromoter for the whey acidic protein gene, tyrosinase related promoters(TRP-1 and TRP-2), DF3 enhancer, TRS (tissue specific regulatorysequences), tyrosine hydroxylase promoter, adipocyte P2 promoter, PEPCKpromoter, CEA promoter, and casein promoter.

Destruction of Fetal Target Cells

Tissue and cellular depletion in the fetus leads to an advantageousenvironment for regeneration, by providing space in the tissue for newcell growth, production of growth factors favorable to cellular growth,protection from injury by the immune system, and protection frominfection.

Ideally, the injury should be as early as possible without causing thedeath of the fetus. The fetal tissue should be subjected to injury andcellular depletion at a stage of gestation between the initial fetaldevelopment of the tissue and the birth of the host animal. Theselective and conditional destruction of fetal target cells can beaccomplished in various ways. For example, one could take advantage ofthe increased sensitivity of hematopoietic cells to radiation andadminister localized radiation to the fetuses before infusing thereplacement cells. Alternately, several proteins have been associatedwith early hematopoietic stem cells and progenitor cells, such as CD34(23) and c-kit receptor (24). Transgenic mammals could be produced thatexpress a suicide gene under the CD34 or c-kit receptor promoter. If atransgenic mammal is used with the suicide gene expressed universally,the prodrug can be delivered with liposomes containing stem cell factor(specific for the c-kit receptor) or an antibody to CD34.

Preferably the fetus is a chimeric animal with one transgenic and onenormal parent. Typically, the male parent is a transgenic animal thatexpresses a suicide gene. A “suicide gene” is a gene that encodes anenzyme that converts a nontoxic prodrug into an active toxin that causesapoptosis. The suicide gene is typically viral or prokaryotic. Examplesof suitable suicide genes include, but are not limited to, thymidinekinase (either wild-type or comprising a mutation), cytosine deaminase,carboxylesterase, carboxypeptidase, deoxycytidine kinase,nitroreductase, guanosine xanthin phosphoribosyltransferase, purinenucleoside phosphorylase, and thymidine phosphorylase. In the absence ofthe prodrug, expression of the suicide gene preferably has no toxic orother adverse effects on normal cellular metabolism (15).

Various prodrugs are available for selectively killing kill that expressthe corresponding suicide gene product. The products of the suicidegenes act on a prodrug, converting them into a toxin. In the absence ofthe suicide gene product, the prodrug is relatively innocuous. Examplesof prodrugs for thymidine kinase include gancyclovir, 6-methoxypurinearabinoside, and (E)-5-(2-bromovinyl)-2′ deoxyuridine. An Example of aprodrug for cytosine deaminase is 5-fluorocytosine. An example of aprodrug for carboxylesterase is irinotecan. A prodrug forcarboxypeptidase is4-([2-chloroethyl][2-mesyloethyl]amino)benzyol-L-glutamic acid. Examplesof prodrugs for deoxycytidine kinase include 4-ipomeanol cytosinearabinoside and fludarabine. Examples of prodrugs for guanosine-xanthinphosphoribosyl transferase include 6-thioxanthine and 6-thioguanine. Anexample of a prodrug for nitroreductase is5-aziridin-2,4-dinitrobenzamidine. An example of a prodrug for purinenucleoside phosphorylase is 6-methylpurine deoxyribonucleoside. Examplesof prodrugs for thymidine phosphorylase include 5′-deoxy-5-fluorouridineand 1-(tetrahydrofuryl)-5-fluorouracil.

The preferred embodiment for selectively and conditionally injuringfetal cells is to administer a prodrug to a fetal transgenic non-humanmammal in a manner that injures cells of the target tissue withoutcausing injury to the maternal host or to the foreign replacement cellsregenerating the tissue. The transgenic mammal expresses a suicidetransgene product in cells of the target. When exposed to thecorresponding prodrug, these cells die.

The presence of the suicide gene product in the appropriate fetal tissuecan be established by sampling tissues from fetal transgenic mammals andanalyzing them with Western blots with specific antibodies.

If desired, subpopulations of target cells such as hematopoietic cellscan be targeted. For example, B cells can be targeted in transgenicanimals carrying a suicide transgene under the control of a CD19promoter or by delivering the prodrug with immunoliposomes directed tothe CD19 antigen. Nerve tissue can be removed surgically. Transgenicmammals Transgenic mammals using a promoter or immunoliposomes specificfor fetal nerve tissue, such as calbindin-D28k, can be used (26).

Myoblasts differentiate into smooth muscle, skeletal muscle or cardiacmuscle (cardiomyocytes). Selective destruction of the respective musclecan be accomplished by targeting specific promoters or antigens, such assarcolemmal extracellular ATPase for smooth muscle (27), ryanodinereceptor in skeletal muscle (28), and ventricular myosin light chain forcardiac muscle (29). Epithelial cells can be targeted based on therespective cytokeratins. Fetal retinal pigment epithelium can be removedsurgically or targeted based on related proteins. Native pancreaticislets could be depleted with streptozotocin (18), or the beta cellscould be targeted based on specific proteins, such as the insulinpromoter. Endothelial cells can be targeted based on the promoter forvon Willibrand's factor or antibodies to specific adhesion moleculessuch as ICAM-2 (30). Renal cells can be ablated with a suicide geneunder a kidney specific promoter or antibodies to renal antigens. Nativebreast ductal cells can be targeted using breast specific promoters andantigens, such as 24-17.2 (31). Host intestinal epithelial cells in thebase of the intestinal crypts are radiosensitive and express uniqueantigens (32).

The suicide gene product preferably is expressed in the appropriatetissue of the fetal host mammal at a level sufficient to be sensitive toa prodrug. The optimal dose of a prodrug depends on the prodrug, theparticular suicide gene, the level of suicide gene expression in thetarget cells, zygosity of the fetal mammal, route of administration, andplacental transport. Optimal doses can readily be determined byadministering increasing doses of the prodrug to the pregnant mammal,assessing the effect on the target cells using standard pathologymethods and assessing the effect on fetal survival using ultrasound andpathology techniques. The optimal dose would produce significant cellinjury (>20%) with a minimum of fetal deaths (<15%). For example, whenpregnant mice carrying fetal mice heterozygous for thymidine kinaseunder the albumin promoter (expressed in the liver) were injected withincreasing doses of gancyclovir, it was found that an intravenousinjection of 25 mg/kg produced significant hepatocyte necrosis but thefetal pups survived. At 50 mg/kg and higher, the fetal mice died.

The prodrug is administered to the fetal host at a gestation period whenthe target tissue is present and suicide gene is expressed in the fetalhost, generally between the end of the first trimester and before birthof the animal. If the tissue-specificity is determined by thetissue-specific promoter, the timing may also be influenced byproduction of the transgene products. For example, the α-fetoproteinpromoter is expressed in fetal hepatocytes. So the appropriate prodrugwould deplete a portion of the fetal hepatocytes. AFP is also expressedin the yolk sac (19). Therefore, it would be preferable to delayadministration of the prodrug until the yolk sac becomes insignificant,at the end of the first trimester.

Prodrugs that cross the placental barrier can be administered to thematernal host. Such drugs include gancyclovir (thymidine kinase),fludarabine (deoxycytidine kinase), and1-(tetrahydrofuryl)-5-fluorouracil (thymidine phosphorylase). Theplacental transport of the proposed prodrug can readily be determinedempirically. Following systemic administration to the maternal host,fetal tissues can be sampled for levels of the prodrug. With transgenicanimals, the prodrug is administered systemically and the cellularinjury in the fetal tissues can be assessed by standard histology. Adose response study establishes the optimal dose of prodrug, whichproduces significant tissue injury with maximum survival of the fetuses.

Prodrugs that do not cross the placental bather can be administereddirectly to the fetal host. Methods for administering drugs to fetusesare known in the art. For pigs, for example, the optimal dose ofgancyclovir is about 25 mg/kg (ranging from about 5 mg/kg to about 1000mg/kg), and is administered at approximately 45 days gestation (fromabout 25 to 114 days).

The specificity of destroying the fetal target cells without injuringthe foreign replacement cells or maternal host cells also can beaccomplished in other ways. In another embodiment, transgenic fetalanimals comprising a suicide transgene under the control of a universalpromoter express the suicide gene product in all tissues. Suitableuniversal promoters include the MoMLV LTR, RSV LTR, Friend MuLv LTR,adenovirus promoter, neomycin phosphotransferase promoter/enhancer, lateparvovirus promoter, Herpes TK promoter, SV40 promoter, metallothionenIIa gene enhancer/promoter, cytomegalovirus immediate early promoter,and cytomegalovirus immediate late promoter. Optionally, an induciblepromoter can be used, e.g., a metallothionein gene promoter. Liposomescomprising a tissue-specific targeting ligand specific for the fetaltarget cell or immunoliposomes comprising a surface antibody thatspecifically binds to an antigen on a target cell are then injected intothe fetal animal. The methods for production of liposomes andimmunoliposomes containing prodrug are described in the literature (36,17).

Specificity for the target tissue is determined by the ligand orantibody on the surface. The specific antibody or ligand would bedetermined by the system used. For example, to deplete fetalhepatocytes, liposomes would be produced carrying a ligand for theasialoglycoprotein receptor expressed on hepatocytes. The fetal tissueswould be killed, but the other fetal tissues would not be affectedbecause they do not have the receptor. The regenerating cells would notbe affected because they would not contain the suicide gene product. Thematernal hepatocytes would not be affected because of limited transportof the liposomes across the placenta.

The liposomes would not affect the foreign replacement cells that do notexpress the suicide gene product. The liposomes would not significantlyaffect the maternal host because either the maternal host does notexpress the suicide gene product or the liposomes do not cross theplacental membrane into the maternal circulation.

The appropriate prodrug can be contained within the liposomes orimmunoliposomes or can be administered separately. The optimal dose of aparticular prodrug and the optimal dose of liposomes or immunoliposomescan be determined as described above.

For example, to replace pig hepatocytes with human hepatocytes,transgenic pigs can comprise a suicide gene, such as thymidine kinase,under the control of a universal promoter, such as a CMV promoter. Thetransgenic pigs (either female, male, or both) are bred. Liposomes areproduced containing gancyclovir (optimal dose about 50 μM, range about 2to about 500 μM). In another embodiment, transgenic pigs comprise acytosine deaminase suicide gene under the control of the universalpromoter. The liposomes or immunoliposomes contain 5-fluorocytosine(about 2 to about 500 μM). The liposomes or immunoliposomes can have agalactosylated surface that containscholesten-5-yloxy-N-(4-((1-imino-2-B-d-thiolgalactos-ylethyl)amino)butyl)formamide(Gal-C4-Chol), which binds specifically to the asialoglycoproteinreceptor expressed on the surface of hepatocytes (17). Alternatively anantibody that specifically binds to an asialoglycoprotein receptor canbe linked to a liposome, for example using a polyethylene glycol link.The liposomes or immunoliposomes are injected into the fetal pig atabout 45 days (range about 25 to about 114 days) or at the equivalentgestation period for other non-human mammals. Foreign replacement cellscan be injected simultaneously or at a time shortly before or after theliposomes or immunoliposomes.

When a transgenic male is bred with a non-transgenic female, the suicidegene product is expressed only in the fetuses. It is not expressed inthe maternal host or in the regenerating foreign replacement cells.Administration of a prodrug leads to destruction of the select fetaltissue cells.

In another embodiment, the transgenic mammals express the suicide geneproduct in most or all tissues. The specificity for the target cells isaccomplished by placing the prodrug in the fetal mammal in a manner thatit enters primarily the target cells.

In another embodiment, transgenic non-human mammals express the suicidegene product in most or all tissues. Specificity for the target cellscan be accomplished by providing the prodrug directly to the tissuecontaining the target cells. For example, if the fetal hosts expressthymidine kinase in all cells, including hepatocytes, the prodruggancyclovir can be injected under ultrasound guidance into the liver,where the prodrug would kill the hepatocytes.

There are also methods for selectively and conditionally injuring anddestroying fetal cells and tissue that do not use transgenic animals. Inone implementation, the fetal cells would be transfected with genetherapy using a vector carrying the suicide gene. This could be a viralvector such as a defective adenovirus, or a non-viral vector such asliposomes or naked DNA.

The fetal tissues could be surgically removed. For example, much of theliver could be removed by surgically resection. This would lead toregeneration of the liver with the new cells.

Fetal cells also can be depleted by chemical means. For example, whenstreptozotocin has been injected into fetal rabbits and sheep, itdestroys the pancreatic islets, leading to diabetes in the fetal animals(18). However, it has relatively little effect on the maternal islets.

Foreign Replacement Cells

“Foreign replacement cells” are defined as cells capable ofproliferating and, optionally, differentiating into mature cells withina tissue to regenerate the tissue. Such cells include differentiatedcells, progenitor cells, tissue-specific stem cells, multipotent stemcells, and omnipotent stem cells. The cells may be from the same speciesor from a different species (xenogeneic) and may be primary cells orcells of a cell line. Cells from the same species can be used to producefactors in the chimeric animal that have therapeutic value. For example,pig hepatocytes can be transfected with human factor VIII. When thesecells replace the native pig hepatocytes, the chimeric animal wouldproduce human factor VIII. Chimeric animals comprising cells from adifferent species can be used as models for human physiology anddisease, for the development of new drugs and vaccines, for theproduction of human factors for therapeutic use, and to provide cellsand organs for transplantation. Foreign replacement cells may or may notbe genetically modified.

Tissue based stem cells have the ability to proliferate anddifferentiate into the corresponding tissue cells. For example,pancreatic duct cells can differentiate into islets of Langerhans.Hepatic oval cells can differentiate into hepatocytes.

Adult stem cells and certain tissue-based stem cells are known to haveplasticity, being capable of differentiating into other types of cells.For example, hematopoietic stem cells can differentiate into endothelialcells, neurons, glia, hepatocytes, cardiomyocytes, renal tubular cells,pulmonary epithelium, intestinal cells, skin epithelium, bone,cartilage, muscle, fat, and brain (20, 21).

Embryonic stem cells have the ability to proliferate indefinitely anddifferentiate into any tissue. Either the embryonic stem cells, celllines produced from embryonic stem cells, or progenitor cells derivedfrom the embryonic stem cells or cell lines would regenerate the injuredfetal tissue.

Typically, foreign replacement cells are injected into the fetus in asterile manner using technology known to those active in the field. Thecells should be of sufficient number to regenerate the tissue. Generallymore cells are more effective than fewer cells. The cells capable ofregenerating the tissue should be present at a time following the injuryand destruction of target host cells. They may be injected before theinjury, provided that they survive until after the injury occurs.Optimally, the cells should be placed in the fetal host before the birthof the host mammal, in order to benefit from the protection provided tothe fetus by the maternal host and immune hyporeactivity to the foreignreplacement cells.

For example, human cells capable of regenerating the liver, such ashepatocytes, liver progenitor cells, or hematopoietic stem cells, can beinjected into fetal pigs. The optimal number of cells injected dependson the source and can be determined empirically using routine screeningmethods. For pigs, optimally the cells are injected at 52 days gestationor seven days after the prodrug (range 25 to 114 days gestation). Theoptimal dose of human hepatocytes is 5×10⁶/pig (range 1×10⁵ to 5×10⁷cells/pig). The optimal number of liver stem cells is 5×10⁵/pig (range 1to 5×10⁷/pig) (16). Bone marrow and cord blood provides a source ofpluripotential progenitor cells that can differentiate into hepatocytes.The optimal dose of cord blood is 2.5×10⁷ nucleated cells/pig (range1×10⁶ to 10⁸). The cells preferably are injected using a steriletechnique. Ultrasound guidance can be used. The injection technology isgenerally known to those familiar with the art.

Methods for Separating and Enriching a Foreign Replacement CellPopulation from a Mixture of Foreign Replacement Cells and Host MammalCells

The invention provides methods for efficiently removing host cells froma cell suspension containing both host cells and foreign replacementcells. For example, if human hepatocytes were grown within a fetaltransgenic non-human mammal containing a suicide gene product, it wouldbe advantageous to separate the non-human mammal cells from the humancells. The human cells could then be used, for example, fortransplantation into patients. Human cells also can be used in devices;for example, human hepatocytes can be used artificial livers, providingtemporary life support for patients in liver failure.

In one embodiment, a mixture of cells from a tissue of a transgenicnon-human mammal containing a suicide gene product and non-transgenicforeign replacement cells is placed in a liquid medium containing aprodrug for that activates the suicide gene. In the presence of thesuicide transgene product, the transgenic cells die while the foreignreplacement cells remain viable. For example, if the suicide transgeneis thymidine kinase, gancyclovir or a gancyclovir analogue is added tothe culture medium (optimally at about 20 μM, ranging from about 1 μM toabout 10 μM). After a period of time (e.g., from about 1 to about 20days, preferably about 7 days), the non-human mammalian cells would dieout, leaving a suspension enriched for foreign replacement cells. Deadnon-human mammalian cells and cell fragments can be separated from liveforeign replacement cells using established technology well known tothose skilled in the field.

In another embodiment, non-human mammalian host cells contain atransgene that facilitates separation of these cells from the foreignreplacement cells. For example, the transgene can encode greenfluorescent protein (GFP). The non-human mammalian cells can then beseparated from the foreign replacement cells that do not express GFP byfluorescence activated cell sorting.

In another embodiment, a mixture of cells from a transgenic mammalcontaining a suicide gene product and non-transgenic foreign replacementcells is cultured in medium containing a prodrug for the suicide geneproduct. The transgenic cells die, while the foreign replacement cellsremain viable. For example, gancyclovir or an analogue can be added to aculture of a mixture of human hepatocytes transgenic pig cellscomprising thymidine kinase (optimally 100 mg/l or 4 mM gancyclovir,range 2 to 1000 mg/l). After a period of time (optimally 7 days, range 1to 20 days), the pig cells die, leaving a suspension enriched for humanhepatocytes.

To purify foreign replacement cells from native host cells, the suicidegene product could either be expressed in select cells under a specificpromoter or expressed globally under a universal promoter. However,global expression would permit more thorough purification. In the aboveexample, pig cells with the suicide gene under an albumin promoter wouldpermit elimination of the native hepatocytes, the primary contaminatingcell. A suicide gene controlled by a universal promoter, however, wouldhelp eliminate other cells as well, including endothelial cells, Kupfercells, etc.

There are multiple methods for separating dead cells from viable cells.If viable cells adhere to the culture flask, dead cells can be removedby removing the media and washing the adherent cells with media. Deadcells also can be removed by centrifuging the mixture over a densitygradient.

Other transgenes can also facilitate depletion of the native cells fromthe foreign replacement cells, such as new antigens allowing forantibody mediated separation, ligands for receptors, and proteins thatchelate iron, allowing for magnetic removal.

A mixture of cells procured from the chimeric host mammal to determinethe presence and amount of host mammalian cells remaining afterpurification procedures. Cells from the transgenic mammal contain aunique transgene in the genome, allowing for sensitive polymerase chainreaction assays using primers specific for the transgene. Routine andquantitative PCR assays are well known to those in the field. Expressionof transgene products permits easy quantitation of the remaining cells.For example, the expression of green fluorescent protein in the hostmammalian cells permits quantitation of the host cells by flowcytometry.

Normal Maternal Host Comprising a Fetus Having a Targeted CellularInjury

The invention provides a normal maternal host comprising a fetalnon-human mammal in which one or more targeted cellular injuries hasbeen induced. The cellular injury in the fetus is conditional and can beinduced at the discretion of the user. This provides an efficient andcost effective bioreactor for growing foreign replacement cells,including transfected cells for production of therapeutic factors,models for the study of disease and development of drugs and vaccines,and a source of cells and organs for transplantation.

The combination of normal maternal host and fetal non-human mammalcomprising such a cellular injury provides advantages for growingforeign replacement cells within the fetal host, including a structuralframework for regeneration with foreign replacement cells with space forthe foreign replacement cells, appropriate growth factors, physiologicsupport from the maternal host, protection from immune rejection, and asterile environment. The host species should be fertile and able toreproduce. Injury to target tissue in the fetal non-human mammal can becontrolled by the user and can be specific for select cells in the fetusbut not in the maternal host or regenerating cells. Such a fetus cancontinue to live for an extended period within the uterus of thematernal host mammal. The fetus preferably is receptive to the placementof regenerating cells during fetal development.

All patents, patent applications, and references cited in thisdisclosure are expressly incorporated herein by reference in theirentireties. The above disclosure generally describes the presentinvention. A more complete understanding can be obtained by reference tothe following specific examples, which are provided for purposes ofillustration only and are not intended to limit the scope of theinvention.

Example 1 Growth of Human Hepatocytes in Fetal Pigs

These studies demonstrate that human hepatocytes survive in fetal pigsand produce human liver proteins.

Pig livers can provide temporary support for patients with acute liverfailure. Because many proteins produced by the liver are speciesspecific, however, these xenografts might not be adequate for long-termsupport. To produce chimeric pig livers repopulated in part with humanhepatocytes, fetal pigs were infused with human hepatocytes.

Human hepatocytes were injected into the livers of pre-immune fetal pigs(45 days gestation) using ultrasound guidance. Frozen and thawed humanhepatocytes were infused into 8 piglets (2 or 5×10⁶ cells) from twolitters. The two piglets of another litter were infused with humanhepatocytes transformed with SV40 (10×10⁶ cells). After farrowing, afourth litter was euthanized at 5 days and the tissues analyzed. Pigletsof the first three litters were followed by testing their sera for humanliver proteins. Alpha-1 antitrypsin (α1-antitrypsin) was detected withWestern Blots. Serum amyloid A (SAA) was quantified by ELISA. Tissueswere evaluated using PCR to detect class I human leukocyte antigen inthe major histocompatibility complex and immunoperoxidase staining usinga monoclonal antibody to detect human albumin.

Two injected piglets from the first litter were stillborn. Theirtissues, however, contained human DNA. The other three injected pigswere healthy, as were the five chimeric pigs from the other two litters.All showed evidence of human hepatocytes, with levels of human SAA at0.1% to 0.9% of normal human levels. Alpha-1 antitrypsin was assessed inthe serum of three pigs, which had levels of α1-antitrypsin between 1and 22% of normal human levels.

The spleens of all five pigs from the first litter contained human DNA.Immunoperoxidase staining of tissues of the euthanized pigs for humanalbumin showed individual human hepatocytes in the spleen and scatteredlobules in the liver. The lobules had a normal sinusoidal architecture.SAA was detected in the sera of the other five pigs for up to 90 days(as long as these pigs were followed).

These studies demonstrate that human hepatocytes survive in fetal pigsand produce human liver proteins. Based on the cells observed in theliver and spleen, it is estimated that the human hepatocytes expanded29- to 90-fold. Apparently, the native pig hepatocytes limited theengraftment of the human cells, because engraftment of human cellsamounted to less than 3% of the pig liver.

Example 2 Growth of Human Hepatocytes from Marrow or Cord Blood Cells inFetal Pigs

This example demonstrates that both human bone marrow and cord blood areappropriate sources for hepatocytes.

Hematopoetic stem cells demonstrate plasticity, with the ability todifferentiate into somatic cells. In this study, human marrow or cordblood cells were infused into fetal pigs. The resulting chimeric pigswere assessed for evidence of human hepatocytes and serum proteinssecreted by the human hepatocytes.

Human marrow cells (1.5 to 3×10⁷/pig) or cord blood cells(1−1.5×10⁷/pig) were injected into the livers of pre-immune fetal pigs(45 days gestation) using ultrasound guidance. The marrow was depletedof 75% of the CD4⁺ and CD8⁺T cells. The pigs were delivered by Cesareansection at term. At 1 to 2 weeks of age, serum was evaluated for humanserum amyloid A (hSAA) and human albumin (hAlb) using an ELISA.Paraffin-embedded sections of liver were stained by immunoperoxidase forhuman albumin using an immunoperoxidase method.

Serum was available from 17 pigs injected with human marrow and 13 pigsinjected with cord blood cells. Fourteen of the marrow-injected pigswere considered chimeric. The concentration of hSAA was 62+/−152 ng/mlfor the marrow-injected pigs. The human albumin in the marrow-injectedpigs was present at an average concentration of 7+/−8 μg/ml.Immunohistochemistry of liver sections demonstrated lobules ofhepatocytes expressing human albumin, with apparent cellular expansion.The hepatocytes showed normal sinusoidal architecture. The periportalregions, however, did not contain hepatocytes expressing human albumin.The area of tissue stained was less than 3% of the total. The pigsinjected with human cord blood cells showed similar results.

Example 3 Expression of a Suicide Gene Product in a Cell Line andSensitivity to a Prodrug

This example shows that selective expression of a suicide gene productand selective killing of target cells can be controlled by atissue-specific promoter.

Liver cell lines were transfected with suicide genes controlled byeither an albumin or an α-fetoprotein promoter. Albumin andα-fetoprotein are expressed primarily in hepatocytes, with α-fetoproteinexpressed more in immature hepatocytes and liver carcinoma lines.

Mouse and human liver cell lines were transfected with constructscontaining delta thymidine kinase under the control of either a porcinealbumin or α-fetoprotein (AFP) promoter. The mouse cell line (TIB73) isa line of mature hepatocytes in which albumin is expressed. The humancell line (Huh-7) is a line of hepatocellular carcinoma cells thatexpress predominantly AFP. The transfected cells were tested forthymidine kinase by Western blot.

To assess the function of mutated thymidine kinase, Huh-7 cells werestably transfected with AlbxTK. Transfected cells were selected using200 μg/ml Zeocin. Transfected cells as well as untransfected cells wereplated in duplicate in 24-well plates at a 40 percent confluence ratio.Gancyclovir was added to the cells in the culture media at differentconcentrations (4 μM, 8 μM, and 16 μM). After 5 days, the cells weretrypsinized, washed twice in PBS, and stained with propidium iodine forFACS analysis.

As shown in FIG. 1, thymidine kinase was expressed by both the mousehepatocyte line (TIB73) and the human hepatocellular carcinoma line(Huh-7). Expression with the albumin promoter was greater with themature hepatocyte line, while the expression with the α-fetoproteinpromoter was greater in the hepatocellular carcinoma line. While PCRanalysis demonstrated the presence of the transgene in non-liver lines(fibroblasts, renal tubular epithelial line PK-15) transfected withthese plasmids, expression of thymidine kinase was not observed byWestern blot.

Incubation with 8 or 16 μM gancyclovir caused significantly more celldeath at 5 days. The Huh-7 cells were entirely killed at 7 days.Gancyclovir had only a minimal effect on non-transfected cells.

Example 4 Selective Destruction of Fetal Hepatocytes in Using a SuicideGene Under Control of a Tissue-Specific Promoter

This example demonstrates that selective tissue injury of the targetcells can be produced in a fetal host using a suicide transgene underthe control of a target tissue-specific promoter without significantlyaffecting the maternal host. It also demonstrates production of fertilemales that carry the transgene and that the prodrug gancyclovir crossesthe placental barrier to cause injury in the fetal target tissues.

Transgenic mice were produced with the FVB strain containing the mutatedthymidine kinase transgene under the albumin promoter. The males fromthe F2 generation were fertile and homozygous. These males were bredwith non-transgenic FVB mice. At 14 days gestation, gancyclovir (0 to100 mg/kg body weight) was injected intravenously into the pregnantmice. Tissues from the mothers and fetuses were analyzed by histology.

No injury was observed in the fetuses or mothers injected with vehicle.At 25 mg/kg, the fetal mice had partial necrosis of the hepatocytes. At50 and 100 mg/kg, there was massive hepatic necrosis. There was noobservable injury to the hepatocytes of the mother or to the non-livertissues in the fetal mice at any dose.

Example 5 Immunoliposome Delivery of Prodrug to Cell Lines Containing aSuicide Gene Product and an Appropriate Surface Antigen

One implementation of the invention uses transgenic host mammals thatexpress the suicide gene product in all tissues. Tissue-specificity isprovided by immunoliposomes carrying an antibody or liposomes with aligand specific for a select receptor. This example demonstrates therelative specificity provided by immunoliposomes.

Immunoliposomes were produced with mouse anti-pig Class I SLA antigen onthe surface and containing gancyclovir at concentrations from 0 to 100μM. The immunoliposomes were incubated with cell cultures of PK-15 (pigrenal tubular cell line) transfected with thymidine kinase under thecontrol of a universal promoter or with Huh-7 (human hepatocyte line)with thymidine kinase under the control of an α-fetoprotein promoter.

The immunoliposomes killed cells from the pig cell line, which expressesclass I SLA antigen, in a dose response manner. The immunoliposomes didnot significantly affect the human cell line, which contained thesuicide gene product but not the class I SLA antigen. Immunoliposomeswithout gancyclovir did not affect either cell line.

Example 6 Development of Transgenic Pigs Containing a Suicide Gene

This example demonstrates the construction of herds of transgenic pigscontaining suicide genes.

Briefly, fibroblasts from 35-day-old fetal pigs were cultured and thentransfected with a suicide transgene (either a mutated thymidine kinaseor cytosine deaminase) using electroporation. Colchicine was added toarrest the transfected fibroblasts in G2/M phase. Swine oocytes werecultured, and the polar bodies were removed. Transfected fibroblastswere inserted into oocytes using a micromanipulator. Followingactivation, embryos were implanted into surrogate sows at estrus. Thelitters were monitored by ultrasound. At term, the pigs were deliveredby Caesarean section.

Cord blood from the transgenic pigs were analyzed for the presence ofthe transgene using PCR. The transgenic pigs were bred, and the F1 andF2 fetal pigs were analyzed by Western blot for the presence of thesuicide gene. Sensitivities to the corresponding prodrug (gancyclovirfor thymidine kinase, 5-fluorocytosine for cytosine deaminase) wereassessed in other sows carrying the suicide gene.

The genetic constructs were used for the suicide transgenes are shown inFIG. 2. The nucleotide sequence of a porcine albumin promoter is shownSEQ ID NO:1. xTK is a mutated version of a Herpes simplex virus (HSV)thymidine kinase gene that was mutated by two rounds of site-directedmutagenesis, resulting in the replacement of adenosine for cytosine atnucleotides 130 and 180, to prevent male sterility. The nucleotidereplacements resulted in a codon change such that a leucine replaces amethionine. These changes do not affect the enzymatic activity of thegene, because the catalytic pocket is located far away from themutations. The four constructs also contain green fluorescent promoterunder a universal promoter. This aids in the analysis of chimericanimals and in the separation of native pig cells from foreignreplacement cells.

Three transgenic male pigs with the Alb-xTK transgene were delivered.Western blot analysis of fetal F1 pigs demonstrated the presence ofthymidine kinase in the liver. Transgenic pigs were delivered and raisedwith the AFP-cytosine deaminase transgene, CMV-xTK transgene, orCMV-cytosine deaminase transgene. Western blot analysis confirmedexpression of the product of the AFP promoter-controlled transgene inthe fetal liver and of CMV promoter-controlled transgene in multipletissues (liver, lung, heart, brain).

The four herds of transgenic pigs are the first transgenic pigscontaining suicide genes. This is the first use of porcinetissue-specific promoters (albumin and α-fetoprotein), and the firstpigs containing the mutated thymidine gene.

Example 7 Enhanced Engraftment of Human Hepatocytes in Fetal Pigs withSuicide Transgenes after Exposure to a Prodrug

The fetal environment is most suitable for growing foreign replacementcells. Growth factors are abundant, the environment is sterile, andrejection of the foreign replacement cells does not occur. The maternalhost provides physiologic support. However, the native cells severelylimit the amount of engraftment. To overcome this problem, native fetalpig liver cells are killed by administering the prodrug gancyclovir tothe sow. Because the sow does not contain the transgene, the sow livercells are not affected. The prodrug crosses the placental membrane andkills a portion of the pig liver cells in those fetal pigs containingthe transgene. This is followed by infusion of human cord blood cells,which provides a source of progenitor cells to regenerate the fetal pigliver with human hepatocytes.

A non-transgenic sow is bred with a boar that is hemizygous for amutated thymidine kinase transgene under the control of an albuminpromoter. Pregnancy is confirmed with ultrasound and gancyclovir (100mg/kg, i.v.) is administered at 40 days gestation. A laparotomy isperformed at 45 days. Each pig is infused with 10×10⁶ human cord bloodcells. The piglets are delivered at term by Caesarean section. Blood isdrawn from each fetal pig and analyzed for the presence of the suicidetransgene using PCR. Serum is tested for the presence of human liverproteins, including albumin, α1-antitrypsin, and serum amyloid A.Sections of liver are frozen and stained for human albumin.

Piglets without the thymidine kinase transgene express levels of humanalbumin, α1-antitrypsin inhibitor and serum amyloid A at less than 1% oflevels seen in humans. Piglets with the thymidine kinase suicidetransgene have levels of the three proteins at 20 to 37% of normal humanvalues. Sections of liver from pigs without the suicide transgene showscattered lobules of human hepatocytes (e.g., less than 2% of the totalliver). Liver sections from pigs with the thymidine kinase suicide geneshow large areas engrafted with human hepatocytes (e.g., an average of25% of the total liver).

Thus, extensive engraftment of the fetal pig liver with humanhepatocytes can be achieved by exposing fetal pigs having a thymidinekinase suicide transgene to the prodrug gancyclovir, whereas pigswithout the suicide transgene show very limited engraftment.

Example 8 Conditional and Selective Destruction of Host Cells after anAnimal is Born

Transgenic boars are produced that express the suicide gene thymidinekinase under the albumin promoter. Accordingly, the suicide gene ispreferentially expressed in hepatocytes, particularly in juvenile andadult pigs.

The transgenic boars (preferably homozygous for the suicide gene) arebred with wild type gilts or sows. During fetal development (30 to 90days gestation, preferably 40 to 60 days), the fetal pigs are injectedwith progenitor or stem cells that develop into human hepatocytes. Theymay be human hepatocytes from isolated human cadavers. One to 20 millionhepatocytes (preferably 2 to 5 million) are injected into the liver orabdomen of each fetal pig.

After birth of the pig, the serum or liver may be examined for evidenceof human cells. The prodrug for thymidine kinase, such as gancyclovir isadministered to the pig, at a dose known to destroy a portion of the ofpig hepatocytes. Following recovery, the prodrug may be administered onmultiple occasions.

Example 9 Conditional and Selective Destruction of Host Cells after anAnimal is Born

Transgenic mice are produced that express the thymidine kinase geneunder the albumin promoter. The male mice are bred with wild typefemales. Human hepatocytes (2×10⁶ per fetal mouse) are injected at 17days gestation, just prior to delivery. At 14 days of age, the mice areinjected with gancyclovir (50 mg/kg) on days 1 and 3 or with thevehicle. Thirty days later, the mice are sacrificed. The mice treatedwith gancyclovir show substantially greater engraftment with humanhepatocytes than the controls not treated with gancyclovir.

Example 10 Replacement of Host Cells with Transfected Cells to ProduceModels of Human Disease

Conditional SCID Pig with Transgenic Pigs Expressing Thymidine KinaseUnder the jak3 Kinase Promoter.

Mice and humans with X-linked immune deficiency have nearly absent Tlymphocytes and deficient B lymphocytes. In both species, the X-SCID isassociated with a mutation of the jak3 tyrosine kinase. Sequencing ofthe promoter shows a high degree of conservation between humans and mice(32). In contrast to typical SCID models, the conditional SCID pigs areimmune competent until treated with the prodrug. They are therefore mucheasier to produce. The conditional SCID pig is very useful for manydifferent purposes, including development of stem cell transplants,development of surrogate tolerogenesis, or cancer research. For example,the conditional SCID pigs can be made immune deficient and transplantedwith a human tumor line that metastasizes to the liver. New proceduresfor ablation of metastatic tumors can then be tested.

The porcine promoter for jak3 is isolated from a cDNA library andexpanded using primers flanking the 5′ and 3′ regions. The porcinesequence is determined. The albumin promoter in the pTracerpAlbxTKplasmid is replaced with the porcine jak3 promoter. Transgenic pigs areproduced and screened for the transgene in the bone marrow. Bone marrowis further screened for mRNA by RT-PCR.

The transgenic herd is expanded through cloning or breeding.

Transgenic pigs are treated weekly with Gancyclovir. The peripheralblood is evaluated at weekly intervals for T cells, B cells andimmunoglobulins.

Example 11 Replacement of Host Cells with Transfected Cells to ProduceModels of Human Disease

Conditional Knockout and Replacement of Endothelial Cells withTransgenic Pigs Expressing Thymidine Kinase Under the von WillebrandPromoter.

The primary target of rejection in organ xenografts is the endothelium.Most work with transgenic pigs has focused on modifying the endothelium,i.e., complement inhibitors and knockout of the alphaGa1 antigen. Thepace of this research, however, is limited by the time required toproduce a new line of transgenic pigs. In vitro tests can be performedwith transfected cultured endothelium. There are no good in vitro assaysof acute vascular rejection, however. A transgenic pig herd with asuicide gene expressed in the endothelium can be produced, which allowsreplacement of native endothelium with a test endothelium that isgenetically modified. With this system, the hybrid pig organ can betested by transplantation. Multiple modifications can be compared. Whenthe optimal genetic modification is achieved, transgenic pigs forclinical use can be produced. The pace of xenotransplant research can beaccelerated by a factor of 10. These pigs are also valuable for studyinghematopoiesis, atherosclerosis, and to develop better stents.

The porcine von Willebrand promoter is isolated and inserted into thepTracerpAlbxTK plasmid in place of the porcine albumin promoter. Fetalpig fibroblasts are transfected. Transgenic pigs are produced by nucleartransfer. At delivery, cord blood is screened by PCR for the transgenein the DNA. The umbilical cord is infused with trypsin and theendothelial cells cultured. The cells are analyzed by RT-PCR and Westernblot for the production of the TK mRNA and enzyme. The herd is expandedthrough cloning or breeding.

Endothelial progenitor cells are isolated from cord blood ofnon-transgenic pigs using Meltenyi beads and antibodies to porcine CD34or CD31. The progenitor cells are transfected with yellow gfp under auniversal CMV promoter. The transfected cells are infused into fetalpigs. The pigs are subsequently administered the prodrug Gancyclovir todeplete a population of native endothelial cells. Pigs are euthanized.Lung and liver sections are cannulated, washed and perfused with trypsinto isolate endothelial cells. The relative number of cells with yellowfluorescence is quantified by flow cytometry and compared with similarengraftment studies performed with nontransgenic pigs.

Example 12 Replacement of Host Cells with Transfected Cells to ProduceModels of Human Disease

Replacement of native animal cells with cells from a human withcongenital abnormality.

To produce an animal model of Gaucher's disease, hepatocytes from ahuman cadaver are isolated and injected into fetal transgenic pigsexpressing a suicide gene, such as thymidine kinase under the albuminpromoter. Later, the native pig hepatocytes are depleted by administereda sublethal dose of the prodrug gancyclovir. This can be done onmultiple occasions.

Example 13 Replacement of Host Cells with Transfected Cells to ProduceModels of Human Disease

Replacement with Cells in which the Test Gene has been Inserted BetweenCre-Lox or FLP Codes by the Appropriate Recombinase Enzyme.

To produce more predictable outcomes, the replacement cells can comefrom a herd of transgenic animals with the Cre-Lox or FLP marker genes.The appropriate recombinase enzyme can replace the DNA between themarkers with the desired transgene. Herds can then be readily producedthat compare different genes for the development of the disease. Forexample, different apolipoproteins can be produced by different herdsand the development of coronary atherosclerosis determined.

All patents, patent applications, and scientific references cited inthis disclosure are expressly incorporated by reference in theirentireties.

REFERENCES

-   1. McCune, J., Kaneshima, H., Krowka, J., Namikawa, R., Outzen, H.,    Peault, B., Rabin, L., Shih, C. C., Yee, E., Lieberman, M., and et    al. The SCID-hu mouse: a small animal model for HIV infection and    pathogenesis. [Review]. Annu Rev Immunol 9, 399-429. 1991.-   2. Aaberge, I. S., Michaelsen, T. E., Rolstad, A. K., Groeng, E. C.,    Solberg, P., and Lovik, M. SCID-Hu mice immunized with a    pneumococcal vaccine produce specific human antibodies and show    increased resistance to infection. Infect. Immun. 60(10), 4146-4153.    1992.-   3. Cao, T. and Leroux-Roels, G. Antigen-specific T cell responses in    human peripheral blood leucocyte (hu-PBL)-mouse chimera conditioned    with radiation and an antibody directed against the mouse IL-2    receptor beta-chain. Clin. Exp. Immunol. 122(1), 117-123. 2000.-   4. Rhim J A, Sandgren E P, Palmiter R D, Brinster R L. Complete    reconstitution of mouse liver with xenogeneic hepatocytes. Proc.    Natl. Acad. Sci. 92:4942-6, 1995.-   5. Sandgren E P, Pahniter R D, Heckel J L, Daugherty C C, Brinster R    L, Degen J L. Complete hepatic regeneration after somatic deletion    of an albumin-plasminogen activator transgene. Cell 66:245-56, 1991.-   6. Mercer, D. F., Schiller, D. E., Elliott, J. F., Douglas, D. N.,    Hao, C., Rinfret, A., Addison, W. R., Fischer, K. P., Churchill, T.    A., Lakey, J. R., Tyrrell, D. L., and Kneteman, N. M. Hepatitis C    virus replication in mice with chimeric human livers. Nat. Med.    7(8), 927-933. 2001.-   7. Braun, K. M., Degen, J. L., and Sandgren, E. P. Hepatocyte    transplantation in a model of toxin-induced liver disease: variable    therapeutic effect during replacement of damaged parenchyma by donor    cells. Nat. Med. 6(3), 320-326. 2000.-   8. Zanjani, E. D., Ascensao, J. L., Flake, A. W., Harrison, M. R.,    and Tavassoli, M. The fetus as an optimal donor and recipient of    hemopoietic stem cells. Bone Marrow Transplant 10 Suppl 1, 107-114.    1992.-   9. Pixley, J. S., Zanjani, E. D., Shaft, D. M., Porada, C., and    MacKintosh, F. R. Prolonged Hematopoietic Chimerism in Normal Mice    Transplanted in utero with Human Hematopoietic Stem Cells.    Pathobiology 66, 230-239. 1998.-   10. Srour, E. F., Zanjani, E. D., Brandt, J. E., Leemhuis, T.,    Briddell, R. A., Heerema, N. A., and Hoffman, R. Sustained human    hematopoiesis in sheep transplanted in utero during early gestation    with fractionated adult human bone marrow cells. Blood 79,    1404-1412. 1992.-   11. Beschorner, W. E., Qian, Z., Mattei, P., Hess, A. D.,    Colombani, P. M., Sciscione, A. C., Khouzami, A., Blakemore, K. J.,    and Burdick, J. F. Induction of human chimerism and functional    suppressor cells in fetal pigs: feasibility of surrogate    tolerogenesis for xenotransplantation. Transplant. Proc. 28:648-9,    1996.-   12. Beschorner, W. E., Thompson, S. C., Yang, T., Cederberg, C.,    Fox, I. J., Strom, S. C. Human hepatocytes from fetal pigs infused    with hepatocytes or marrow. Cell Transplant Society, 10^(th)    Anniversary Congress, 2001, p. 83.-   13. Billingham R, Brent L, and Medawar P. Quantitative studies on    tissue transplantation immunity III. Actively acquired tolerance.    1956; Proc R Soc Lond [Biol.]. 238:357-415.-   14. Moore K. L. The Developing Human. Clinically Oriented    Embryology. W.B.Saunders Company. 1973, pp. 1.-   15. Morris J. C., Touraine R, Wildner O., and Blaese, R. M. Suicide    Genes: Gene Therapy Applications Using Enzyme/Prodrug Strategies. In    Friedmann T., Ed. The Development of Human Gene Therapy. Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999, pp. 477.-   16. Matsusaka, S., Toyosaka, A., Nakasho, K., Tsujimura, T.,    Sugihara, A., Takanashi, T., Uematsu, K., Terada, N., and    Okamoto, E. The role of oval cells in rat hepatocyte    transplantation. Transplantation 70(3), 441-446. 8-15-2000.-   17. Kawakami, S., Munakata, C., Fumoto, S., Yamashita, F., and    Hashida, M. Novel galactosylated liposomes for hepatocyte-selective    targeting of lipophilic drugs. J. Pharm. Sci. 90(2), 105-113. 2001.-   18. Hay, W. W., Jr. and Meznarich, H. K. Use of fetal streptozotocin    injection to determine the role of normal levels of fetal insulin in    regulating uteroplacental and umbilical glucose exchange. Pediatr.    Res. 24(3), 312-317. 1988.-   19. Yazova, A. K., Goussev, A. I., Poltoranina, V. S., and    Yakimenko, E. F. Human alpha-fetoprotein epitopes as revealed by    monoclonal antibodies. Immunol. Lett. 25(4), 325-330. 1990.-   20. Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E.,    Keene, C. D., Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund,    T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W. C.,    Largaespada, D. A., and Verfaillie, C. M. Pluripotency of    mesenchymal stem cells derived from adult Krause, D. S. Plasticity    of marrow-derived stem cells. Gene Ther. 9: 754-758. 2002.-   21. Ladiges et al, Lab. Anim. Sci. 40:11-15, 1990.-   22. Henry et al. Am. J. Vet. Res. 46: 1714-20, 1985.-   23. Civin, C. I. and Small, D. Purification and expansion of human    hematopoietic stem/progenitor cells. Ann. N.Y. Acad. Sci. 770,    91-98. 1995.-   24. Fujio, K., Evarts, R. P., Hu, Z., Marsden, E. R., and    Thorgeirsson, S. S. Expression of stem cell factor and its receptor,    c-kit, during liver regeneration from putative stem cells in adult    rat. Lab. Invest. 70(4), 511-516. 1994.-   25. Markakis, E. A. and Gage, F. H. Adult-generated neurons in the    dentate gyrus send axonal projections to field CA3 and are    surrounded by synaptic vesicles. J. Comp Neurol. 406(4),    449-460.4-19-1999.-   26. Stout, J. G. and Kirley, T. L. Control of cell membrane    ecto-ATPase by oligomerization state: intermolecular cross-linking    modulates ATPase activity. Biochemistry 35, 8289-8298. 1996.-   27. Moore, R. A., Nguyen, Galceran, Pessah, N., and Allen, D. A    transgenic myogenic cell line lacking ryanodine receptor protein for    homologous expression studies: reconstitution of Ry1R protein and    function. J. Cell Biol. 140(4), 843-851. 1998.-   28. Wobus, A. M., Kaomei, G., Shan, J., Wellner, M. C., Rohwedel,    J., Ji Guanju, Fleischmann, B., Katus, H. A., Hescheler, J., and    Franz, W. M. Retinoic acid accelerates embryonic stem cell-derived    cardiac differentiation and enhances development of ventricular    cardiomyocytes. J. Mol. Cell Cardiol. 29(6), 1525-1539. 1997.-   29. Cowan, P. J., Tsang, D., Pedic, C. M., Abbott, L. R.,    Shinkel, T. A., d'Apice, A. J., and Pearse, M. J. The human ICAM-2    promoter is endothelial cell-specific in vitro and in vivo and    contains critical Spl and GATA binding sites. J. Biol. Chem. 273,    11737-11744. 1998.-   30. Thompson, C. H., Jones, S. L., Whitehead, R. H., and    McKenzie, I. F. A human breast tissue-associated antigen detected by    a monoclonal antibody. J. Natl. Cancer Inst. 70(3), 409-419. 1983.-   31. Brittan, M. and Wright, N. A. Gastrointestinal stem cells. J.    Pathol. 197(4), 492-509. 2002.-   32. Cabot, R. A., Kuhholzer, B., Chan, A. W., Lai, L., Park, K. W.,    Chong, K. Y., Schatten, G., Murphy, C. N., Abeydeera, L. R., Day, B.    N., and Prather, R. S. Transgenic pigs produced using in vitro    matured oocytes infected with a retroviral vector. Anim Biotechnol.    12(2), 205-214. 2001.-   33. Chan, A. W. S., Homan, E. J., Ballon, L. U., Burns, J. C., and    Bremel, R. D. Transgenic cattle produced by reverse transcribed gene    transfer in oocytes. Proc. Natl. Acad. Sci. USA. 95:14028-3, 1998.-   34. Ellison, A. R., Wallace, H., Al-Shawi, R., and Bishop, J. O.    Different transmission rates of herpesvirus thymidine kinase    reporter transgenes from founder male parents and male parents of    subsequent generations. Mol. Reprod. Dev. 41, 425-434. 1995.-   35. Gonzalez-Rothi, R. J., Suarez, S., Hochhaus, G., Schreier, H.,    Lukyanov, A., Derendorf, H., and Costa, T. D. Pulmonary targeting of    liposomal triamcinolone acetonide phosphate. Pharm. Res. 13,    1699-1703. 1996.-   36. Civin, C. I. and Small, D. Purification and expansion of human    hematopoietic stem/progenitor cells. Ann. N.Y. Acad. Sci. 770,    91-98. 1995.-   37. Fujio, K., Evarts, R. P., Hu, Z., Marsden, E. R., and    Thorgeirsson, S. S. Expression of stem cell factor and its receptor,    c-kit, during liver regeneration from putative stem cells in adult    rat. Lab. Invest. 70(4), 511-516. 1994.-   38. Hay, W. W., Jr. and Meznarich, H. K. Use of fetal streptozotocin    injection to determine the role of normal levels of fetal insulin in    regulating uteroplacental and umbilical glucose exchange. Pediatr.    Res. 24(3); 312-317. 1988.-   39. Yazova, A. K., Goussev, A. I., Poltoranina, V. S., and    Yakimenko, E. F. Human alpha-fetoprotein epitopes as revealed by    monoclonal antibodies. Immunol. Lett. 25(4), 325-330. 1990.-   40. Hay, W. W., Jr. and Meznarich, H. K. Use of fetal streptozotocin    injection to determine the role of normal levels of fetal insulin in    regulating uteroplacental and umbilical glucose exchange. Pediatr.    Res. 24(3), 312-317. 1988.-   41. Yazova, A. K., Goussev, A. I., Poltoranina, V. S., and    Yakimenko, E. F. Human alpha-fetoprotein epitopes as revealed by    monoclonal antibodies. Immunol. Lett. 25(4), 325-330. 1990.

1. A method of obtaining an expanded population of foreign replacementcells, comprising steps of: (a) selectively destroying native cells in atissue of a fetal non-human mammal host, wherein the number of maternalcells of the same tissue is not substantially reduced; (b) implantingforeign replacement cells in the tissue of the fetal non-human mammalhost, whereby the foreign replacement cells replace destroyed nativetissue cells, thereby forming a chimeric tissue comprising an expandedpopulation of foreign replacement cells; and (c) separating the expandedpopulation of foreign replacement cells from the chimeric tissue.
 2. Themethod of claim 1 wherein the step of separating comprises one or moreof fluorescence activated cell sorting, magnetic separation,antibody-mediated separation, ligand-receptor mediated separation, andadministering a prodrug for a suicide gene product.
 3. The method ofclaim 1 wherein the native cells comprise a transgene which encodes aprotein selected from the group consisting of a suicide gene product, afluorescent protein, an antigen, a receptor ligand, and aniron-chelating protein.
 4. The method of claim 3 wherein the transgeneencodes the suicide gene product.
 5. The method of claim 4 wherein afirst population of native cells is selectively destroyed by a firstadministration of a prodrug for the suicide gene product.
 6. The methodof claim 5 further comprising at least one additional administration ofthe prodrug.
 7. The method of claim 4 wherein the suicide gene productis selected from the group consisting of thymidine kinase, mutatedthymidine kinase, cytosine deaminase, carboxylesterase,carboxypeptidase, deoxycytidine kinase, guanosine-xanthinephosphoribosyl transferase, nitroreductase, purine nucleosidephosphorylase, and thymidine phosphorylase.
 8. The method of claim 1wherein the tissue comprises liver, hematopoietic, endothelial, neuralepithelial, retinal, pigment epithelial, myocardial, skeletal muscle,smooth muscle, thymus, pancreas, lung, intestine, kidney, endocrine,cartilage, or bone cells.
 9. The method of claim 1 wherein the fetalnon-human mammal host is selected from the group consisting of aprimate, an artiodactyl, a rodent, a carnivore, and a lagomorph.
 10. Themethod of claim 1 wherein the native cells are destroyed using animmunoliposome.
 11. The method of claim 1 wherein the native cells aredestroyed using a liposome comprising a toxin.
 12. The method of claim11 wherein the liposome comprises a tissue-specific targeting ligand.13. The method of claim 12 wherein the tissue-specific targeting ligandis an antibody.
 14. The method of claim 9 wherein the fetal non-humanmammal is an artiodactyl.
 15. The method of claim 14 wherein theartiodactyl is a pig.
 16. The method of claim 14 wherein the artiodactylis a transgenic artiodactyl.
 17. The method of claim 1 wherein thematernal cells are not transgenic.
 18. The method of claim 3 wherein thetransgene comprises a universal promoter or a cell-specific promoter.19. The method of claim 18 wherein the promoter is the universalpromoter and the universal promoter is selected from the groupconsisting of a MoMLV promoter, an RSV LTR promoter, a Friend MuLv LTRpromoter, an adenovirus promoter, a neomycin phosphotransferasepromoter, a late parvovirus promoter, a Herpes virus thymidine kinase(TK) promoter, an SV40 promoter, a metallothionein promoter, acytomegalovirus (CMV) immediate early promoter, and a CMV immediate latepromoter.
 20. The method of claim 18 wherein the promoter is thecell-specific promoter and the cell-specific promoter is selected fromthe group consisting of an albumin promoter, an α-fetoprotein promoter,a whey acidic protein promoter, a tyrosine-related promoter (TRP), a DF3enhancer promoter, a tyrosine hydroxylase promoter, an adipocyte P2promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, acarcinoembryonic antigen (CEA) promoter, and a casein promoter.
 21. Themethod of claim 1 wherein the tissue is liver.
 22. The method of claim 1wherein the foreign replacement cells are hepatocytes.
 23. The method ofclaim 6 wherein at least one of the at least one additionaladministrations is post-natal.